The dopamine transporter (DAT) is the plasmalemmal membrane protein that mediates the inactivation of released dopamine through its reuptake. DAT is the major molecular target responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. Homologous neurotransmitter transporters for serotonin and norepinephrine are targets for antidepressant medications as well as secondary targets for psychostimulants. The long-term goals of this research are to determine the structural bases of substrate translocation and of efflux induced by psychostimulant substrates such as amphetamine. More specifically, we would like to understand whether DAT-mediated amphetamine-induced dopamine efflux can be blocked while simultaneously preserving dopamine transport and how this would alter the behavioral and neurotoxic effects of amphetamine. For the next grant period, we propose to test the following hypotheses: a) Phosphorylation of specific serines in the N-terminus of DAT switches the transporter from a "resistant" to a "willing" state for amphetamine-induced efflux, b) Phosphorylation of the N-terminus regulates its interaction with the N-terminus of another DAT at an oligomeric interface, c) Distinct kinases are responsible for regulated phosphorylation of the N-terminal serines, d) Amphetamine-induced dopamine-efflux, and not simply blockade of uptake, is responsible for some of the behavioral effects of acute and/or chronic amphetamine in vivo. To test these hypotheses, we propose studies with the following specific aims: 1) To identify the sites of DAT N-terminal phosphorylation and the functional role of phosphorylation. 2) To identify elements in the N-terminus and their interactions at an oligomeric interface with the N-terminus of another DAT and to assess the impact of phosphorylation on these interactions. 3) To identify the kinase or kinases involved in phosphorylating these N-terminal serines. There are numerous advantages to pursuing these studies as part of the proposed Program Project grant. Much of the work carried out during this grant period was conducted as part of collaborations with Aurelio Galli, Ulrik Gether, or Harel Weinstein, and substantial synergies have been achieved through our interactions. Continued building upon these synergies is reflected in the proposed aims of this project, as well as in the interplay of our related aims and, in some cases, related methodologies.